Copyright X)1993, AmericanSocietyforMicrobiology
RNA
Duplex
Unwinding Activity
of Poliovirus
RNA-Dependent
RNA
Polymerase
3DP01
MICHAEL W.
CHO,`*
OLIVER C. RICHARDS,' TATIANA M. DMITRIEVA,2 VADIMAGOL,2'3ANDELLIE EHRENFELD'Department ofMolecularBiologyandBiochemistry, University of California, Irvine, Irvine, Califomnia 92717,1and A. N. BelozerskyInstituteof PhysicalChemicalBiology, MoscowState
University, Moscow119899,2and Instituteof Poliomyelitisand ViralEncephalitides, RussianAcademy ofMedicalSciences, MoscowRegion 142782,3 Russia
Received 28 December 1992/Accepted 13 February 1993
The ability ofhighlypurified preparations of poliovirus RNA-dependentRNApolymerase,3DPo1,tounwind RNAduplex structures wasexamined duringa chainelongation reaction in vitro. Usingan antisense RNA prehybridized toanRNAtemplate,weshow thatpoliovirus polymerase canelongate throughahighly stable RNA duplex of over 1,000 bp. Radiolabeled antisense RNA was displaced from the template during the reaction, and productRNAs whichwereequalinlengthtothetemplatestrandweresynthesized. Unwinding did notoccurinthe absence of chainelongationand didnotrequire hydrolysisof the'y-phosphateof ATP.The rateof elongation through theduplex regionwascomparabletotherateofelongation onthesingle-stranded region of the template. Parallel experiments conducted with avian myeloblastosisvirusreversetranscriptase showed that this enzymewas not able to unwind the RNAduplex, suggesting that strand displacement by poliovirus3DP01 is nota propertysharedbyallpolymerases.
Poliovirus, the prototypic member of the Picomnaviridae
family, has a single-stranded RNA genome of positive
po-larity approximately 7,500nucleotides(nt) long.The genome
is covalently linked to a small viral protein, VPg, at its 5'
terminus(9, 21)andincludesageneticallyencoded
poly(A)
segmentatits 3' terminus(49). Soon after entry of the virus into a host cell, thegenome is released into the cytoplasm
andserves as amRNAtodirect translation ofasingle large polypeptide which is subsequently processed by viral pro-teasesintomatureproteins. Theyinclude structuralproteins
from the P1regionofthegenomeandnonstructuralproteins
from the P2 and P3regionswhich function in theproteolytic
maturation process of viral proteins and in viral genome
replication (for reviews, seereferences 18 and 43).
As a first step in viral replication, the genomic RNA is
copiedtoproduceacomplementaryRNAofnegative polar-ity. Thisreactionis catalyzed by the viralRNA-dependent RNApolymerase,
3DP"",
anenzymebelongingtoa groupof enzymes found only in RNA viruses. Subsequently, this newly synthesized RNA serves as a template to directsynthesisofdaughter genomic RNAs, therebycompletinga round of replication. Despite long-term efforts in several
laboratories, little is known about the biochemical mecha-nism ofRNA-dependent RNA synthesis (for a review, see
reference36).Invirus-infectedcells, RNA synthesis occurs in replication complexes associated with virus-induced membranevesicles ofrough endoplasmic reticulum origin (2,
5). Replication complexes consist of heterogeneous, multi-stranded structures called replicative intermediates (RI) (4,
11,25)in association with an only partially characterized set of RNA replication proteins. RI contains a core RNA molecule of genome length and various numbers of comple-mentary strands of nascent RNA of various lengths. The core structures of RI, when examined after isolation from
* Correspondingauthor.Electronicmail address:
MCHO@UCI.
EDU
virus-infected cells and deproteinization, appearto contain
significant segments of duplex RNA on the basis of their resistanceto digestionby RNase and electron microscopic
studies (38, 42). However, RNA cross-linking studies sug-gest that, in vivo, RI appears to be predominantly
single
strandedwithnascentstrandsduplexedwith theirtemplates onlynearthe
replication
fork(38). 3DP°o
is theonly
protein required for elongation of RNA chains in vitro (24, 47); however,currentmodels propose that, in vivo, 3DPoI must function inconcertwith other viraland/or cellularproteinsto accomplishthe complete reaction. These additional pro-teins have beensuggestedtofunction in avarietyof
activi-tiesthoughtto compose partsofthe RNAreplication reac-tion. Forexample, P2-derived proteins (2Corits precursor
2BC) have beenproposed to be involved in the formation
andmaintenanceofreplication complex-associated membra-nous structures as well as in anchoring replication
com-plexes to these structures (3). The involvement of these proteinsin RNAreplication has beenconfirmed by genetic studiesanalyzingseveralmutantsof2B(17) and 2C(22, 27,
32,45).Inaddition,VPg(3B)oritsprecursor(3AB)has been
proposed to function as aprimerfor the initiation of RNA
polymerization (29, 44).
In order for an RNA strand to serve as template for
multiplerounds ofproduct RNA synthesis, the RNA repli-cativemachinery shouldeither preventformationof exten-sivebasepairingbetween thetemplateRNAand the nascent
complementarystrandorcontainahelicaseactivitywhich is able to unwind RNA duplex subsequent to its formation. Suchamechanism would also be necessarytoreleasenewly
synthesized RNAs to serve as templates for subsequent rounds ofreplication, translation, or packaging. Dmitrieva et al.(7)reported that an RNA helicase activity involved in the
synthesis of encephalomyocarditis virus (EMCV)-specific single-stranded RNAwas observed inrelatively crude rep-lication complexesisolated from virus-infected cells. More
recently, they reportedanATP-and RNA elongation-depen-dent RNAhelicase activityofeitherviral orcellularorigin
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which initially copurified with the EMCV RNA polymerase but was lost upon further purification (8). Identification of the viral 2C protein as a potential nucleoside triphosphate (NTP) binding protein by computer-assisted sequence anal-ysis (12-14), in conjunction with the demonstrated implica-tion of 2C in viral RNA replicaimplica-tion, has led to the suggesimplica-tion that thisprotein performs the RNA helicase activity required for the RNA replication reaction. At present, however, no directbiochemical assay of 2C as an RNA helicase has been reported.
Although genetic studies have provided some hints of the roles of some viral proteins in RNA replication, the molec-ular mechanism of the reaction has not yet been established. The enzyme that catalyzes RNA chain elongation,3DP"',has beenpurifiedboth from virus-infected cells (23, 50) and from
bacteria(30, 33)or insect cells(31) expressing the recombi-nantprotein, and itspropertiesarebeginning to be analyzed
(39).As part of ourlong-term studies of the 3D polymerase protein and its functional activities, we examined the RNA duplex unwinding ability of highly purified recombinant
3DP""
during chain elongation in vitro. We report here that this enzyme can unwind a long stretch of RNA duplex so as todisplace strands for continued polymerization of nascent chains and that the unwinding reaction proceeds in an elongation-dependent, ATP-independent manner requiring noadditionalproteins.MATERIALS AND METHODS
Construction of plasmids. The construction of the plasmids used to generate RNAs to serve as substrates for
3DP"'
unwinding activity assay is diagrammed in Fig. 1. Methods were as described by Sambrook et al. (41). pPOV-3 (16) which contains the3'-terminal segmentofpoliovirus cDNA was digested with BamHI and SalI restriction endonu-cleases.The fragmentcontaining the poliovirus sequence (nt 4600through 3' terminus)wasgelpurified and subsequently ligated into pGEM-4 (Promega) which had been digested with BamHI andSallanddephosphorylatedwith calf
intes-tinalphosphatase. The resulting plasmid, pPV-2C*3D, was usedtogenerateatemplateandaprimerRNA.pPV-2C*3D
wasdigestedwithBglII and SphI, the endswerefilled in with Klenow enzyme, and theplasmidwas religatedtogenerate
pPV-2C*3C*. This plasmid contains poliovirus cDNA se-quence from nt 4600 to 5601 and was used to generate an antisense RNA.
RNA transcription. RNA transcription was performed
with reagents from Promegaaccordingtothe manufacturer's
suggestedconditions with some minor modifications.
Tem-plate RNA (positive polarity) was transcribed from
pPV-2C*3D with SP6 RNApolymerase subsequenttodigestionof the plasmidwithSphI. The primerRNAand the antisense
RNA(negativepolarity)weretranscribed frompPV-2C*3D
and pPV-2C*3C*, respectively, with T7 RNA polymerase subsequent to
digestion
of theplasmids
with ScaI andEcoRI, respectively. To generate 32P-labeled antisense RNA,
[ot-32PjUTP
(Amersham)
was added tothe transcrip-tion reactranscrip-tionto afinalspecific activity
of 12mCi of UTP per mmol. Inaddition,theconcentration of unlabeled UTPwaslowered by 10-fold to0.1 mM,while the concentrations of the other nucleotides were kept at 1 mM.
Subsequent
to transcription at 37°C for 2h, the DNAwasdigested
withRQ1 DNase
(Promega)
at 37°C for 30 min. RNA wasextracted with
phenol-chloroform
andwasprecipitated
with ethanol with 2 M ammonium acetate as a salt to minimizecoprecipitation of
unincorporated
nucleotides. Thepellet
H I4600
glll5601
EcoR I
OR' BA9LIan5HI
tI pPOV-3 rpGEM4 JSalIJr ~~~~~~~~~~~Sph'I
SalIDigest BamHIDigest
I
Sca1 7332 CIP
Sal I Digest BamH IDigest
oigation
N\EcoR I (Apr '~(~ BamHI
{AM
pPV 2C*3D
OR Bg~slII
SphlI
ScaI Bgl Digestof
SphIDigest
Klenow
MEVectoraReligation
\ ~~~~~EcoRI
AApr N BamHI
[image:2.612.322.558.80.436.2]pPV2C3CX
FIG. 1. ConstructionoftheplasmidsusedforsynthesizingRNA transcripts. A BamHI-SalI fragment of pPOV-3 containing the poliovirus sequence was ligated into the multiple cloning site of pGEM-4 to generate pPV2C*3D. pPV2C*3D was subsequently digestedwithBgllland
SphR,
the ends werefilledin,andthevector was religated to generate pPV2C*3C*. The nucleotide numbers indicatedonpPOV-3refertothenucleotideresidues of thepoliovi-rus
genome.
was
resuspended
in TEbuffer(10
mMTris-HCn[pH 8.0],
1 mMEDTA)
andstored at -20wC.RNAsubstrate
preparation.
RNA substrates wereformedby
incubating
mixtures of RNAs at65hC for 30 min in TE buffercontaining
100 mMNaCl.Approximately equal
molar amountsof all three RNAswere derivedby
experimentally
titrating
thehybridization
reactionsothat themobility
of all detectablesingle-stranded
template
RNAwasshiftedtothat ofduplex
RNA on an agarosegel.
No excessprimer
or antisense RNAswereapparentafterstaining
with ethidium bromide. RNA substrates weresubsequently
stored at -20RC. Toprepare
RNAsubstrateswithout detectable lev-els of nucleotidecontamination,
these RNApreparations
were
subjected
to a second ethanolprecipitation
with 2 M ammoniumacetate.To examine the
degree
of basepairing
between thetem-plate
strand and either the antisense strand ortheprimer,
RNAwastreated with RNaseA
(10
,ug/ml)
at30°Cfor 30 min in TE buffercontaining
300 mMNaCl,
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resistant duplexes were analyzed by electrophoresis in an agarose gel.
Purification of poliovirus polymerase 3D. PoliovirusRNA polymerase wasexpressed in Escherichiacoliharboring the plasmid, pEXC-3D, at 30°C, and crude sonicates were prepared as described by Rothstein et al. (40). The lysates were clarified by centrifugation at 100,000 x g, and poly-merase waspurified from the supernatant (S-100),basically as described previously (31). The purification protocol in-cluded 0 to40% saturation ammonium sulfateprecipitation, phosphocellulose (Whatman), Mono-Q (Pharmacia LKB Biotechnology), andPhenyl-Superose (Pharmacia LKB) col-umnchromatography. As afinal step, the enzyme was bound and eluted from apoly(U)-Sepharose 4B (Pharmacia LKB) column (1.5 by 8 cm) in 50 mM Tris-HCl (pH 8.0)-5 mM
3-mercaptoethanol-0.1%
NonidetP-40-10%glycerol,
witha 100-ml gradient from 0.05 M KClto0.4 MKCl.Forall purification steps the presence ofpoliovirus poly-merase was monitored by assaying poly(U) polymerase
activity on a poly(A) template as described by Hey et al. (16). For the final purification step, polymerase puritywas evaluated by analysis of column fractions in a sodium dodecyl sulfate(SDS)-10% polyacrylamide gel and detection ofprotein by silver staining(28).
Poliovirus polymerase and AMV RT assays, and product
analysis.
Approximately0.25 ,ug oftemplate RNA and 30 ng ofpurifiedpoliovirus polymerasewereincubatedat30°C for 1 h under the following conditions unless notedotherwise: 50 mM HEPES(N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid; pH 8.0), 0.5 mM each NTP (Boehringer
Mannheim), 4 mM dithiothreitol,3 mM magnesium acetate, 0.06mMZnCl2,4,ug ofactinomycinDperml, and 0.8 U of RNasin (Promega) per ,ul. In some experiments, 5'-adeny-lylimidodiphosphate (AMP-PNP; Boehringer Mannheim) was used at 0.5 mM. In experiments in which de novo-synthesized RNAwas labeled,
[a-32P]UTP
was added to a finalspecificactivity of4mCi/mmol,andapproximately 1 ,ug of RNAtemplatewasused.Polymerasereactions withavianmyeloblastosis virus (AMV) reverse transcriptase (RT; Promega)wereperformed according to the manufacturer's
suggestedconditions with either 1or0.75 mM deoxyribonu-cleotidesateither 30or42°C. ProductDNAwaslabeled with
[a-32P]dATP
(1.3 mCi/mmol; Amersham). Reaction products wereanalyzed on1% agarose gels under either nondenatur-ing or glyoxal-denaturing conditions as described by Sam-brook etal. (41).RESULTS
RNA substrates for RNA duplex unwinding assays. To examine RNA duplex unwinding activity of poliovirus
RNA-dependent RNA polymerase, we used the strategy previ-ously developed by Dmitrieva et al. (8), applied to a different setofRNAsubstrates. The assay involves a polymerization reaction with an RNA template prehybridized to a segment of complementary, antisense RNA. The antisense RNA serves asanobstacle for apolymerase. RNA duplex
unwind-ingactivity allows displacement of the antisense RNA from thetemplate RNA. The length of the resulting polymerized
product is equal to that of the template RNA if unwinding occurs; a shorterproduct, equal to the length of the unhy-bridized segment of the template, results if the antisense RNAis notunwound and displaced.
Figure 2 showsadiagram of the template RNA (positive
polarity), a template-specific primer RNA (negative
polari-ty),and the antisense RNA (negative polarity), synthesized
i IliC D
I^
IS ..CI
T ....Template .102lt |
-Primer 278nlt
I
[image:3.612.333.535.81.158.2].I
FIG. 2. Diagramof transcripts usedtogenerate RNAsubstrates. Thetemplateand primerRNAsweretranscribed frompPV2C*3D subsequenttodigestion oftheplasmidwithSphI andScaI, respec-tively. The antisenseRNA wastranscribed frompPV2C*3C* sub-sequent todigestion oftheplasmidwithEcoRI.Thelengthsand the polarity of the transcripts are as indicated. El, E, and _ denotepoliovirussequences, poly(A)or poly(U)tract, andvector
sequences,respectively.Thecorresponding regionsof the
poliovi-rus genome areillustratedatthe top.
toproduce substrates for the RNAduplex unwindingassay. The corresponding regions of the poliovirus genome are indicated at the top of the figure. The template RNA is
approximately3,022ntlong, including 2,841ntofpoliovirus
RNAsequences,approximately50ntofpoly(A) tail, plus30 and 101 nt ofvectorsequences at the5' and 3' ends of the RNA,respectively.TheprimerRNAis 278ntlong,
contain-ing 106ntofpoliovirusRNA sequences,approximately50nt
of poly(U) tail, and 122 nt of vector sequences. The
an-tisense RNAis1,047ntlong,including 1,006ntofpoliovirus
RNAsequences,and 21and 20ntofvectorsequencesatthe 5' and 3' ends of theRNA, respectively. Becausethereare 21 ntofvectorsequence atthe 5' terminus of the antisense RNAwhich do not hybridizewith the template, theduplex
formed between thetemplateand the antisense RNA isonly
1,026bp.
Thetemplate-specificprimerRNAwasdesignedtoanneal tothe 3'end of thetemplateandtoextendslightlybeyond it,
since it contained ashort segment(21 nt)of
noncomplemen-tary RNA at its 5' terminus. This prevented the template
from forming a hairpin turn at its own 3' terminus which would allowself-primed RNAsynthesis to occur. We have observed that any template containing a heterogeneous
sequence at its 3' terminus supports primer-independent synthesis thatproduces asnapback product approximately
twice the length of the template (see below). RNAswhich arepolyadenylated attheir 3' termini donotself-prime; the presence of even as few as six heterogeneous nucleotides
following the poly(A) tail, however, results in self-primed
RNAsynthesis bythepoliovirusRNApolymerase (datanot
shown).
Thetranscripts shown in Fig. 2 were annealed in various combinations to produce substrates for the RNA duplex
unwinding assay. A schematic diagram and an ethidium
bromide-stainedgelof these substrates are shown in Fig. 3. Substrate1(lane1)is theRNAtemplatealone.Incubation of thetemplatetogether with primer (substrate 2) resulted in a detectable shift in itsmobility (lane 2), indicating the forma-tion of a partial duplex. The larger duplex region formed between the template and antisense RNA (substrate 3) causedanevengreatermobility shift(lane 3). Substrate 4 is
composedoftemplate,primer, and antisense, and its
mobil-itywasretardedevenmore(lane 4). Substrates 5 and 6 (lanes 5 and 6) are the same as3 and 4 except that the antisense RNA waslabeled with
32p
(indicated byathicker line in thediagram in Fig. 3). These were prepared to allow direct measurement of the displacement of the antisense RNA
Antistrise 047 iit 1-_-.---I
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M 3DPOl
I
3 . _M (Kd)
(Kbp)
3.1
-2.0
-
1.6-I.(.)
0.5
-NI 4 6 NI
66-
a-45- _
[image:4.612.83.277.80.238.2]36- ^_
FIG. 3. Substrates for RNA duplex unwinding assay. Six RNA substrateswere generated, asdiagrammed schematically at the top, by annealingvarious combinations of transcripts shown in Fig. 2. Thesesubstrates were analyzed on a nondenaturing agarose gel and visualized after staining the gel with ethidium bromide. Double-stranded DNA markers areshown in lanes M. The antisense RNA usedtogeneratesubstrates 5 and 6 was labeled with[32P]UTPand isindicated by a thicker line in the diagram.
during the unwinding reaction. Treatment ofsubstrate 6 with RNase A under conditions of high salt specific for the digestion of single-stranded RNAs generated a sharp, RNase-resistant, radiolabeled band of approximately 1,026 bp, indicating stable base pairing between the templateand the antisense RNAs (data not shown). In addition, an ethidiumbromide-stainedband of approximately 300 bpwas observed, representing the template-primer duplex region (datanotshown).
Poliovirus RNApolymerase.We have previously described thepurification andcharacterization of the poliovirus RNA-dependentRNApolymerasefrom clones ofE. coli harboring theplasmid pEXC-3D (31). This plasmidencodes the polio-virus 3CD protein driven bythetrppromoter(37). Induction ofexpressionof3CD results inautocatalyticcleavage by the 3C protease sequence to generate the 3D polymerase pro-tein, identical to thatproduced after infection of cells with poliovirus. SDS-polyacrylamide gel electrophoresis (PAGE) analysisof thepurified proteinused in thesestudies is shown inFig. 4. Nocontaminatingproteinsweredetectedbysilver
staining.
Polioviruspolymerasecanelongate throughalargesegment of RNA duplex. To determine whether an antisense RNA
hybridizedtothetemplatepresentedastable obstacletothe poliovirus polymerase,the RNAsubstrates described inFig. 3 were utilized in a standard polymerase elongation assay containing [32P]UTP, and theproductswereexaminedon a
denaturingagarosegel. Figure5 showsan autoradiogramof the reaction products. When the template alone (substrate 1),withnoprimerandnoantisense RNA,waspresentedas a substrate to the purified poliovirus RNA polymerase, a
predominant product of approximately 6,000 nt was made
(lane 1). Thisproduct is approximatelytwice the lengthof the template and indicated that this template could
self-prime, presumably bycreatinga hairpin structure at the 3' terminus that allowed the synthesis ofa snapback RNA in which thenewly synthesized polynucleotidewascovalently attached to the template. Annealing the antisense RNA to thistemplate (substrate 3)hadno effect onthe self-priming capacity of the template and, more importantly, had no
29-24- a..m
20- N
DF
--FIG. 4. SDS-PAGE analysis of purified poliovirus polymerase. Poliovirus polymerase was purified according to the protocol de-scribed in Materials and Methods. Approximately 300 ng of the purifiedenzyme wassubjectedtoSDS-PAGE(10%acrylamide) and subsequently analyzed by staining the gelwith silver. Molecular weightstandards are shown in lane M, and the dye front is indicated asDF.
effectonthe length of the product(Fig. 5, lane 2), suggesting that the antisense RNA was unwound anddisplaced from the
template allowing polymerization of new product RNA
complementaryto the5' end of the template. Addition ofa primer to these substrates (to generate substrates 2 and 4) produced theproducts shown in Fig. 5, lanes 3 and 4. Some
self-primingstilloccurred,mostlikelyfromexcesstemplates nothybridizedtoprimer.This small amount ofunhybridized templatewould not have been detectableby ethidium bro-mide staining in Fig. 3. The major product, however, was about3,000ntinlength, equaltothelength of the template.
Again,nodifference inproduct lengthwasobserved whether or not antisense RNAwasannealed tothetemplate. These data demonstrate that the poliovirus polymerase elongated
through the region of 1,000 bp presentedby the template-antisense duplexregionand suggest that the enzyme could unwind the duplex. Two small bands migrating near and below1,000nt wereobserved in lanes 2 and 4(Fig. 5).These products appeared when antisense RNA in the absence of
templatewaspresentedassubstrates (datanot
shown),
but theexact nature of these bands hasnotbeen investigated.Parallelexperimentswereconducted with AMV RT
(Fig.
6),
which has beenpreviously
showntobe unabletodisplace complementary RNAfroman RNAtemplate
(6).Thesametemplate-primer substrates, in the presence and absence of antisenseRNA(substrates2and4)wereusedtodirect DNA
synthesiswith AMVRT at30°C.Asa
control,
lane1(Fig. 6)
shows thepredominantly 3,000-nt product of RNA
synthe-sizedbythepolioviruspolymerasewith substrate
2,
asseenbefore in Fig. 5. Small amounts of the
self-primed,
dimer-length product are also visible. RT
produced
afull-length
cDNAof thesame3,000-nt
size(Fig. 6,
lane2);
inaddition,
anotherproductjustover
2,000
ntinlength
is apparent,most--o-
3DPOI
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[image:4.612.370.509.85.315.2]RNASubstrate
e..
1 3 2 4
A?..o
#0_
.... *.f2 2 4 4
---3000nt ---2000nt
-.i.
.71.W.M.W
io
400 iw
.&- --*-lOOOI nt
A 1 2 3 4 A
FIG. 5. RNA synthesis by poliovirus polymerase. RNA sub-strates1, 3, 2,and 4(schematicallyillustrated in thebox)wereused in a3D1') polymerization assayinthe presenceof[32P]UTP. RNA products were denatured with glyoxal and subjected to electro-phoresison anagarosegel,inlanes1to4. Anautoradiogramof the driedgelis shown. The antisense RNA(lanesA; -1,000nt)and the template RNA (not shown; -3,000 nt) were used as mobility markers. The approximate length of the 6,000-nt product was
estimatedfrom itsmobility relativetothemarkers.
likelythe result ofapause (strongstop)by the enzyme due to secondarystructurepresentin thetemplateRNA. When the AMV RT was presented with substrate 4, which con-tained hybridized antisense RNA, the enzyme was
appar-entlyunabletounwind theduplexanddisplacethe antisense
strand, andsynthesis terminated atthe block. This resulted inapredominant cDNA product ofapproximately2,000ntin
length, asseeninFig. 6, lane 3. A shorter product of about 1,000ntwasalsodetected, presumably dueto a RTpause or theantisense RNAasdescribed above(Fig.5, lanes 2 and4).
Identical results were obtained when reactions were per-formedat42°C(thenormal reactioncondition for AMV RT; data not shown), suggesting that the reduced temperature was notthe reason for the inabilityofAMV RTtoelongate
through the RNAduplexregion.
Addition of poliovirus polymerase to the RT reaction did not affect the length of the cDNA product (Fig. 6, lane 4). This result allowed us to exclude the possibility that the RNA unwinding observed in the poliovirus polymerase-catalyzed reaction was due to a contaminating helicase
activityinthe purified enzyme preparation. If a contaminant was unwinding the duplex so as to enable the poliovirus enzyme to elongate to the end of the template, then its unwinding activity would also have allowed RT to synthe-size a full-length product. Thus, the unwinding activity observed in the poliovirus polymerase reaction was inherent inthe enzyme itself.
DisplacementofantisenseRNA from the template. In order todemonstrate directlywhetherpoliovirus polymerase can unwind the antisense RNA from the template, we performed
A 1 2 3 4 A
FIG. 6. DNAsynthesis byAMV RT. RNAsubstrates2 and4 (shown in thebox)wereusedtodirect DNAsynthesis with AMV RT (lanes 2 and 3, respectively). The newly synthesized DNA products werelabeled with[32P]dATP. The reaction productswere denatured with glyoxal and subjected to electrophoresis on an agarose gel. An autoradiogram of the dried gel is shown. Lane 1, RNA products synthesized by poliovirus polymerase with RNA substrate2as acontrol;lane4,polioviruspolymerasewasaddedto the RT reaction shown in lane 3. Approximate lengths of the antisenseRNA(lane A;-1,000nt)andmajor products (-2000 and -3000nt)areindicated.
an antisensedisplacement assay. Inthis assay,weused the substrate (Fig. 3, substrate 6) containing primer and radio-labeled antisense RNA, in a polymerization assay with unlabeled nucleotides. The rationale for this assay is that the antisense RNA, when unwound from the template by an advancing polymerase, would be displaced from the tem-plate, and theradioactivityin thesubstrate woulddisappear
with time. Concomitantly, a corresponding signal would appearasfreeantisense RNA. Sampleswereremoved from apolymerization reactionatvarious timesduring the course of the assay, and theproductswereanalyzedon a
nondena-turingagarosegel.Figure7Ashowsanautoradiogramof the gel. Reaction products from 0, 15, 30, 60, 90, and 120 min of incubation are shown in lanes 1 to 6, respectively. As
expected, the radioactivity in the substrate gradually
de-creased, accompanied byan accumulation ofaband
comi-grating with the antisense RNA. The kinetics of antisense
displacement correlated with thekinetics of RNA
polymer-ization in the same reaction. The displaced product in-creasedlinearlywithtimeduring the first 60 min but reached a plateau thereafter. This shows directly that the antisense strandwasunwound andreleased from the template. Omis-sion of the poliovirus polymerase from the reaction incu-bated for 120 min resulted inastable RNAduplex,withno
displacement of the antisense RNA (lane 7). Inaddition to free antisense RNA, a minor product that migrated more
slowlythan the substrate appearedduringthecourseof the
reaction, butnotin reactions in which the polymerase was
1-2
4=-3DPOI
RT
RNA
r2 -L.I
i'4-I"
---600()nt
4".am --*--3000nt
-.*-
-IOOOnt
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[image:5.612.101.253.81.320.2] [image:5.612.351.516.81.319.2]RNA DUPLEX UNWINDING BY 3DP0' 3015
I,
.i
+3DP0'6
-0' 15' 30' 60' 90'120'120' -+ ++ +- 3DPOIRT .5\ 2
-
).-anOnam m
_o
_
A 1_2 4 5 6 7A A 1 2 3
FIG. 7. Antisense RNA displacement assay. RNA substrate 6 (Fig. 3) containing a radiolabeled antisense RNA was used in polymerizationassays, with eitherpoliovirus polymerase or AMV RT. (A) Kinetic analysis of RNA duplex unwinding activity by poliovirus polymerase. Reaction products from the indicated times of incubation with(lanes 1 to 6) or without3DP"" (lane 7, 120min) wereanalyzed on a nondenaturing agarose gel. An autoradiogram of thedried gel isshown. The antisense RNA (lanes A) is used as a mobility marker. The positions of the RNA substrate, reaction intermediate, and displaced antisense RNA are indicated on the rightside of the panel. (B) Products of RT reaction (1-h incubation at30°C) in whicheither RT alone (lane 1), RT and poliovirus3DP°o (lane 2),orpoliovirus 3DP°' alone (lane 3) was used. The positions of theRNAsubstrate and reaction product are indicated on the right sideof the panel.
omitted. This product comigrated with the fully duplexed final product of the polymerization reaction, and thus it appears tobe an intermediate in which the polymerase has synthesized up to the 5' terminus of the antisense RNA. During the polymerization reactions, not all of the templates were transcribed. In general, approximately 50% of the templates were converted to product during 120 min of reaction. Somewhat less than50% of the antisense RNA was recovered as a discrete band on the gel; some of the
antisense RNAoccurs asduplex intermediate asdescribed
above, and it ispossiblethatsomemightbedegraded.
Theantisense RNA migrates astwobands in a
nondena-turingagarosegel(Fig.7A, laneA).Thedistribution of RNA in each band was concentration dependent with higher
concentration favoring the more slowly migrating band. Heating the sample immediately prior to electrophoresis reduced the proportion which migrated more slowly. The upperband thus appearstobealooselybase-paired dimer;it
migrates just above a 1,018-bp DNA marker and it was sensitivetodigestionwith RNase A under saltconditions in which stable duplexes were resistant and single-stranded
RNAswere digested(datanotshown).
Antisense displacementwas also examined after reverse
transcriptionofthesamesubstrate with AMV RT
(Fig. 7B).
Lane 1 shows that although the amount of label from the substrate band decreased, no antisense RNAwas released
duringthereversetranscription. Instead, aband of
approx-imately 1,000 bpwasgeneratedpresumably bythe RNase H
activity of RT which hydrolyzed the RNA strand of the RNA-DNA duplex formed after cDNA
synthesis, thereby
releasing the remaining template-antisense RNA duplex.The same product profile was observed when
poliovirus
polymerase was added to the RT (lane2),
alsosuggesting
A 1 2 3 4 5 6 7 8 9 A
FIG. 8. NTP requirements of unwinding activity by poliovirus polymerase.RNAsubstrate 6 (Fig. 3) was used in an RNA unwind-ingassaywith either no NTP(lane 1), all four NTPs (lane 2), or three NTPsexcludingATP,CTP,GTP, or UTP (lanes 3 to 6, respective-ly).Products were analyzed on anondenaturing agarosegel; next cametheautoradiographicexposureof thedried gel. Lane 7 shows
areaction in which AMP-PNP alone was present. CTvP,GTP, and UTP(lane 8)or all four NTPs (lane 9)were addedin addition to AMP-PNP. TheantisenseRNA(lanes A) is usedas amarker. The bracketshowsasmearof reaction intermediates which accumulated inlane3.
that the poliovirus polymerase preparation does not have a
contaminatingRNAhelicase. Lane 3 shows poliovirus poly-merase alone under DNA synthesis reaction conditions for RT asanegative control.Asimilar pattern was attained from reaction in which neither RT nor poliovirus polymerase was present(data notshown).
Unwinding depends on chain elongation but not ATP
hy-drolysis.
To determine whether duplex unwinding required RNA polymerization, strand displacement was measured with substrate 6(Fig. 3) under conditions which precluded RNAsynthesis. When all ribonucleotides were omitted from the reaction, no unwinding occurred (Fig. 8, lane 1).Sup-plementationwithall four ribonucleotides restored
unwind-ingand antisensedisplacement(lane 2),butadditionof only three did not (lanes 3 to 6). The absence of unwinding activityinthese reactionsadditionallysupportstheprevious
conclusion that contaminating helicase activity was not present in our purified enzyme preparation. Some activity
wasobserved in reactions which lacked onlyATP(lane 3);
thiswas likely due to minor contaminations of ATP in the mixture ofCTP,GTP, and UTP.Reducingthe NTP concen-trationin the reaction from 500to25 ,uM eliminated the low level ofactivity observed in the absence of ATP (data not
shown). Ribonucleotide titrations showed that RNA
poly-merization and duplexunwinding occurred, albeit less
effi-ciently,with NTP concentrationsaslow as5 ,uM,which is
100-foldlowerthan thestandardreaction conditions.
Thus,
only low levels of ATP contamination in the other NTPpreparationsat500,uMcouldsupplysufficientATPforsome activity to be observed. Under the conditions of
limiting
reaction, asmear ofintermediate RNAproducts
were seen to accumulate above thetemplateband(lane 3, brackets).
The experiments described above demonstrated that
hy-drolysis of ATP or other NTPswas notsufficient for poly-merase-catalyzed RNAunwinding.
To determine whetherhydrolysis of the y-phosphate of ATP was
required
inaddition to chain
elongation,
anonhydrolyzable
analog
ofAl
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[image:6.612.360.533.81.241.2] [image:6.612.64.303.81.245.2]ATP,
AMP-PNPwassubstituted for ATP in the reaction. Nounwinding
occurred in the presence of AMP-PNP alone(Fig.
8,
lane7); however,
fullactivity
was restored whenCTP,
GTP,
and UTP(lane 8)
orwhen all four NTPs(lane 9)
wereadded to the AMP-PNP reaction
(compare
to lane2).
Un-winding
assaysdoneat25,uMNTPtoexclude thepossibility
that the
unwinding activity
wasdueto minor contaminants of ATP inCTP, GTP, UTP,
and AMP-PNPpreparations
also revealed that AMP-PNP could substitute for ATPduring
unwinding activity (data
notshown).
Theseexperiments
show thatwhile RNAchain
elongation
isrequired
for RNAduplex unwinding activity, hydrolysis
of ATP is notre-quired.
DISCUSSION
Many
viruses that infectbacterial, animal,
orplant
cells contain theirgenetic
information in asingle-stranded
RNA genomeofpositive
polarity.
These viruses encodeanRNA-dependent
RNApolymerase
whichcatalyzes
theunique
reaction of RNA
synthesis
from asingle-stranded
RNAtemplate. Replication
of viral RNA takesplace
intwostages:synthesis
ofacomplementary
(negative polarity)
RNAwith the viral genomeastemplate,
and thensynthesis
of progenyvirus
genomic
RNAwith thenegative-strand
RNA astem-plate.
In the case ofpoliovirus
RNAreplication,
RNAsynthesis
occurs viapartially
double-stranded structurescalled
replicative
intermediates. To prevent accumulation of dead endproducts
in whichnewly synthesized
RNAstrands areextensively hybridized
to theirtemplates,
the nascentstrand must be
prevented
from basepairing
with thetem-plate
orbedisplaced by
anunwinding activity. Unwinding
ofdouble-stranded DNA or RNA is an essential process in many cellular
functions, including replication,
recombina-tion, repair, transcriprecombina-tion,
mRNAsplicing,
and initiation of translation of mRNAs. This task isgenerally accomplished
by NTP-dependent
DNAor RNAhelicases whichtranslo-cate
along
onestrand ofduplex
DNAorRNAcoupled
tothehydrolysis
ofATP,
resulting
inunwinding
of theduplex.
Inthis
study
weinvestigated
the RNAduplex unwinding
ability
ofpoliovirus
RNA-dependent
RNApolymerase,
3DP0,during
a chainelongation
reaction in vitro. We showed thatpoliovirus
polymerase
canelongate through
ahighly
stable RNAduplex
of over1,000 bp.
This wasachieved
by unwinding
of theduplex
anddisplacing
the antisenseRNAfrom thetemplate.
Theunwinding activity
is an inherent property of the enzyme. It did not occurin the absence ofconcomitant RNA chainelongation,
andit didnotrequire
hydrolysis
of the-y-phosphate
of ATP.Therefore,theactivity
exhibitedby poliovirus polymerase
is different fromthoseoftrueDNAorRNAhelicases whichmustharvest the energy from
hydrolysis
ofnucleotides in orderto drive the reaction(10).
Since RNAduplex unwinding activityof3DP"l
is
dependent
onelongation,
it isimplied
that thedirection ofunwinding
occursfrom 3' to5'with respect tothetemplatestrand. Onthe basis ofourobservation thatonlyverysmall amounts of reaction intermediates accumulated during the course of the antisense
displacement
assay (Fig. 7A), the rateofelongation
throughtheduplexregion does not appearto differ much from that occurring on the single-stranded
region
of thetemplate.
This conclusion is supported by the fact thatantisense RNAsweredisplaced
within 15 min of the reaction(Fig.
7A,
lane2),
which is consistent with theelongation
rate reported in previous studies (31). AlthoughRNA
duplex
unwinding by
3DP01
occursquite efficiently,
therole, if any, of this activity in RNA replication in vivo has notbeen demonstrated.
It was previously reported that partially purified EMCV RNApolymerase preparations from virus-infected cell ex-tracts were able to displace complementary RNA from a template in a reaction that was both elongation and ATP dependent (8). This unwinding activity was not consideredto be an inherent property of the EMCV polymerase since the activity was lost upon further purification of the enzyme. No restoration of the unwinding activity, however, was achieved byreconstituting different fractions from the puri-fication procedure. Therefore, one possible explanation for the apparent discrepancy with the findings reported in this study could be that the unwinding, but not the elongation property of the EMCV polymerase was inactivated during purification of the enzyme. Alternatively, the properties of the poliovirus and EMCV polymerases may be inherently different. For example, EMCV polymerase may be less processivethan thepoliovirusenzymeand therefore may be unable toelongate through a duplex region of thetemplate. To circumvent this problem, EMCV may have evolved to utilize an RNA helicase(either viral or cellular) as part of its replication machinery.
Most positive-stranded RNA viruses of both plants and animals encodeaprotein which contains nucleotide binding motifs and which appears to be involved in viral RNA replication(12-14).Theseproteinsappeartobehomologous tothe 2Cproteinsof thepicornaviruses,andthey have been postulated to function as NTP-dependent RNA helicases. Thecylindrical inclusion (CI) protein of plum pox potyvirus (apositive-strand RNA plant virus), which contains the NTP binding motif, has been demonstrated to have a nucleic acid-stimulated ATPase activity and an ATP-dependent RNAhelicaseactivity (19, 20). AlthoughCIproteinhas been
postulated to be involved in RNA replication, the exact biochemical function(s) of this protein has not been eluci-dated. From site-directed mutagenesis studies (27, 45), the nucleotide binding motifs of poliovirus 2C have been
sug-gestedtobeimportantin viral RNAreplication.At present, however, no biochemical evidence that 2C is an
ATP-dependentRNAhelicase has been demonstrated.
Hayes and Buck (15) have reported previously that puri-fied RNA-dependentRNApolymerase of cucumber mosaic viruscatalyzesthecomplete replication of cucumber mosaic virus genomic RNA (i.e., synthesis of progeny plus strand froma plus-strand template provided in the reaction). Cu-cumber mosaic virus polymerase contains two virus-en-codedpolypeptides(laand2a)and onehostpolypeptide. On the basis of the observation that the la subunit of the
polymerase contains sequence motifs characteristic of nu-cleic acid helicases, the investigators suggested that the
complete replicationof viral RNA waspossible because of a helicase activity present in the purified polymerase. They also suggested that 2C has an analogous function in the processofpoliovirus RNA replication. To date, no in vitro systemhas beendeveloped which can catalyze the complete replication of poliovirus RNA. Since we have shown that duplex RNA does not
impose
any noticeable resistance to chain elongation by3DP",
we suggest that the inability to reconstitute an in vitro system which can catalyze the complete replication reaction is likely because of problems in initiation rather than elongation.The observation that the poliovirus polymerase by itself can unwind an RNA duplex during the elongation reaction doesnotexclude the possibility that 2C is an RNA helicase. The molecular mechanism ofinitiation of RNA synthesis is
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still unknown; for example, it has not been determined what components are involved, what the order of reactions is, or how the synthesis of negative strands differs from that of the positive strands. Since 3DPo1 is the only protein known to be required and sufficient for the elongation reaction in vitro, it is possible that 2C is involved in RNA unwinding during the initiation of RNA synthesis and 3DPo1 unwinds RNA duplex during chain elongation.
The RNA duplex unwinding activity exhibited by poliovi-rus polymerase during chain elongation does not appear to be a common feature shared by all polymerases, since AMV RT did not unwind the same substrate used for poliovirus polymerase. Different polymerase systems appear to have evolved alternate mechanisms to solve the problem of du-plex formation between the template and the nascent strand during nucleic acid synthesis. Several reports have shown that the bacteriophage +6, which has three segments of double-stranded RNA as its genome, transcribes semicon-servatively such that one of the parental strands of each duplex is displaced by a newly made strand (46, 48). This scheme appears to be quite similar to that of the elongation reaction by polioviruspolymerase described in this study in which nascent strands form duplex structures with the template while displacing the prehybridized antisense RNA. It is not known whether the 46 polymerase is sufficient for this strand displacement or requires another auxiliary pro-tein(s). In the case of the transcription reactioncatalyzed by eukaryotic RNA polymerase II, a nascent RNA strand is prevented from hybridizing to the template DNA by the polymerase because of the enzyme's ability to bind the nascenttranscript (35). According to the model, the nascent RNA enters a product binding site on the protein surface while the transcription bubble advances along with the polymerase which thereby prevents formation of product-template duplex. In the case of bacteriophage Q3, the template RNA rather than the polymerase is responsible for the prevention of duplex RNA. According to the proposed mechanism, both the template strand and the nascent RNA product are displaced fromintermolecular base pair interac-tions in favor of the formation of local intramolecular base pairing (26, 34). This hypothesis has been supported by the observation that RNAs that form less stable intramolecular structures have a greater tendency to form extended RNA duplexes duringreplication, resulting in reduced synthesis of new RNA strands (1).
As expected from previous data (6), RT did not elongate through the region of the template which was hybridized to acomplementary RNA strand. During a retrovirus replica-tion cycle, a complementary DNA strand is synthesized from the viral genomic RNA, generating an RNA-DNA duplex. Subsequently, the RNA strand of the duplex is digested by the RNase Hactivity of RT, andgenomic DNA is synthesized. RT can unwind and displace RNase H hydrolysis products from a DNA template (6), although it was unable to unwind RNA-RNA duplex. Thus, separation ofproduct fromtemplate occursby thecombined action of RNase H and an unwinding-likeactivity of RT.
This study demonstrates that poliovirusRNApolymerase possesses an unwinding activity whichdisplacesextensively
base-paired segments of RNA from its template preceding
progression of a replication fork, allowing basepairing of the nascent strand to the template. The role of this activity
during replication of poliovirus RNA is unknown; it may removesecondary structure in thetemplateorit mayunwind duplex segments of different strands of RNA. Clearly, the release of template from product RNA strands to allow
furtherrounds ofsynthesisappears to be a requirement for thereplicationreaction.
ACKNOWLEDGMENTS
This work was supported by grant AI 17386 from the National Institutes of Health. T.M.D. was a recipient of a short-term Fogarty InternationalFellowship.
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